Individuals allergic to domestic mites are in general sensitive to mites of genus Dermatophagoides, being D. pteronyssinus and D. farinae the most prevalent species worldwide. Additionally, in tropical and sub-tropical regions the presence of B. tropicalis is very abundant, being predominant sometimes in the population of mites present in house dust. In these regions, individuals allergic to mites are sensitive mostly to D. pteronyssinus and B. tropicalis (1).
About twenty allergens have been identified among domestic mites. However, only some of those have shown high reactivity frequency in the allergic population and some show cross-reactivity. Amongst B. tropicalis allergens, Blo t5 shows the highest reactivity frequency, Blo t12 and Blo t 13 show lower frequency, but induce an intense IgE in many of the sensitive individuals (2, 3).
Others as Der p1 and Der p2 are the most important D. Pteronyssinus, and between 50% and 100% of allergic individuals have IgE specific against these two allergens.
Another D. Pteronyssinus important allergen is Der p7, being estimated that 50% of individuals allergic to domestic mites shows IgE against this allergen. Allergens of mite groups 8 and 10, such as glutathione S transferase (GST) and tropomyosin, respectively, are of particular importance due to the significant crossed-reactivity observed with homologous derivate from different mite species and other organisms (4).
An allergen must produce cross-linking of IgE antibodies linked to the effector cells surface in order to induce cell activation and inflammatory response, requiring this process at least two IgE epitopes on the allergen surface. The IgE antibodies of an allergic patient may recognize continuous epitopes or discontinuous epitopes. Cross-linking of IgE antibodies on mast cells surface induce immediate release of biologically active mediators such as histamine and leukotrienes.
Decrease of allergenic capability can be achieved by intervention of IgE epitopes through mechanisms such as point mutation, amino acid deletion and disturbance of the natural molecular bending. Reordering amino acid segments conforming the primary sequence of the allergen also modifies the allergenic capability.
In some studies fusion or hybrid proteins have been generated to provide a better allergen-specific immunotherapy. Fusion proteins have been generated by using several portions of different allergens linked to form a unique molecule, intended to obtain vaccines poorly reactive to IgE antibody, which decreases the anaphylaxis risks, and to deliver to the patient only those allergens involved in development of allergic response (5).
A number of patents is known in this field related to pollen allergens (U.S. Pat. No. 7,862,828) and to domestic mites, as shown in documents US20080274059; US20110052640; US20070065468; US20060233839; US20090130130 and WO2007140505.
In fact, document U.S. Pat. No. 7,862,828 describes a method to prepare fusion allergens, consisting of two or more recombinant polypeptides from grass pollen, to be used as immunotherapeutic agent comprising: (a) to provide a polynucleotide sequence which codifies the fusion allergen, (b) to introduce said polynucleotide sequence into the host cell, (c) to grow the host cell to express the sequence, (d) to recover the fusion allergen expressed from the grown cell, (e) to assay the fusion allergen as a candidate for immunotherapy, by delivering the allergen to a test animal and to select those inducing the strongest immune response and generation of IgE blocking antibodies in comparison to that obtained with the individual components.
On this case, the polypeptide has at least two different allergenic proteins or their fragments. The hybrid polypeptide, the polynucleotide and the cell are useful for drug preparation for treatment of allergenic disorders or for prophylactic vaccination. Among allergenic sources for allergenic proteins, grass pollen, mites, bee venom or animal hair allergens may be the most important. Specific examples of allergenic proteins are group 1, group 2, group 4, group 5, group 6, group 11, group 12 and group 13 of allergens from the most important grass pollen, Der p 1 and Der p 2 (mites), phospholipase from bee venom, and Fel d 1 (cat).
On the other hand, document US20080274059 reveals fusion protein comprising a group 1 allergen and a group 2 allergen from genus Dermatophagoides fused at group 1 allergen N— o C—, where they may be Der p 1 or proDer p 1, and, from group 2, where they may be Der p 2. This document includes claims for the nucleic acid to develop the fusion protein, the expression cassette, the vector containing this cassette and a cell of any type including cassette or expression vector, and a vaccine composition including the fusion protein on a pharmaceutically acceptable support. The fusion protein is applied to prevent or treat allergic reactions to mites. The expression cell may be Escherichia coli, Pichia pastoris or Saccharomyces cerevisiae. 
The United States Patent Application 20110052640 reports a hypoallergenic hybrid polypeptide comprising an amino acid sequence of at least 50 amino acids of a sequence selected from group 1 and 2 allergens from dust mites (Dermatophagoides pteronyssinus), and where a linkage epitope to IgE antibody of said allergen is deleted. Included are either the polypeptide having 70% sequence identity, the corresponding polynucleotide, the vector, the expression host cell including the polynucleotide, the method to produce the polypeptide and the pharmaceutical composition that comprises the polypeptide. The group 1 allergen is Der p 1, and that of group 2 is Der p 2. The obtained polypeptide keeps immunogenic capabilities, being particularly useful for allergy treatment. Additionally, production methods for these polypeptides in heterologous expression systems and efficient purification methods are described
Patent US20070065468 reports a product for specific reduction of immunoglobulin E (IgE) as well as the allergic reaction, specifically a chimeric polypeptide having at least two mite allergens. It includes amino acid sequence for those allergens such as Der p 1, Der p 2 and Blo t 5. Allergens can be expressed in Escherichia coli or CHO—K1.
Patent US20060233839 describes a recombinant protein with an allergen derived from Der p1 where the protein has a reduced allergenic activity, important in relation to the allergen in natural state and comprising three mutation sites. In addition to Der p 1 allergen, ProDer p 1 and Der p 3 with ProDer p 3 and PreProDer p 3 are included. Claims include the nucleic acid molecule, the expression vector, the cell transformed for production of recombinant protein, and the immunological composition used for patient treatment.
Document US20090130130 refers to an isolated polypeptide, derivative or fragment composed by (a) a polypeptide, its derivate isoform, or a fragment thereof, comprising a non-helicoidal mutant of at least an allergen from group 5 mites and, (b) a polypeptide, its derivate isoform, or a fragment thereof comprising at least a substituted, added or deleted amino acid, or at least a chemical modification, where the polypeptide exhibits reduction in reactivity equal to or higher than IgE from the natural polypeptide, in subjects allergic to at least one allergen from group 5 mites. This polypeptide from the group 5 allergens may be Blo t 5, Der p 5, Der f 5, or Der m 5. Claims include nucleic acids sequences, a vector/host cell comprising nucleic acid molecules, a pharmaceutical preparation including the polypeptide or a vaccine.
Patent WO2007140505 reports a hypoallergenic protein comprising an allergen fused to or conjugated with at least a second non-allergenic protein or a fragment thereof. Included are claims for nucleic acid molecules encoding a hypoallergenic molecule or fusion protein, the vector, the host cell, an antibody addressed against the hypoallergenic or fusion protein, and the vaccine which may comprise it. The protein is derivate from Phl p 5, Fel d 1, Der p 2, Der p 7, Der p 21, Clone 30, Alt a 1, Par j 1, Ole e 1, Fel d 2, Can f 1, Can f 1, Art v 1, Amb a1, Alt a 2 or Alt a 6 having a terminal blunt in C- or N-terminus, and exhibiting reduced capability to IgE in comparison to native Phl p 5. The second protein is a viral, bacterial, fungal, or protozoan protein. For example, it is a virus capsid protein, preferably from picornaviridae.
Additionally, it is known that Linhart and collaborators (6) built, by PCR-based recombination, a fusion molecule composed by the most important allergens from timothy grass, in the order Phl p6, Phl p2, Phl p5 y Phl p1, which was expressed in E. coli as a 79 kDa protein. This molecule demonstrated to be useful for immunotherapy since it induced lymphoproliferation in a degree similar to that induced by a equimolar mix of the individual allergens in an model of mice allergic to pollen, induced Thl and IgG profile cytokine production, able to block degranulation of mast cells incubated in the presence of the allergen.
The concept of fusion proteins has been also explored with mite allergens; a fusion protein composed by Der p1 and Der p2 portions showed lower IgE reactivity in comparison to native allergens and may induce mice to produce blocking IgG, which suggests their usefulness in immunotherapy (7).
Recently, Bussieres L. and collaborators (8) built several hybrid proteins composed by Der p1, Der p2 portions and a Der p1 precursor (pro-Der p1), showing that they are able to induce basophile degranulation in individuals allergic to domestic mites, and by “immunoblotting” with polyclonal and monoclonal antibodies, the conservation of B epitopes of native allergens was demonstrated.
Nevertheless, there is a non-satisfied need for vaccines based on recombinant molecules that contain several antigenic regions from mites common in tropical and subtropical areas, which may be suitable alternatives to immunotherapy schemes currently known, which are based on application of natural extracts from complete allergens, and which may be able to decrease the anaphylaxis risk and to improve the treatment efficacy.